CIMAC - pixel clustering, cell clustering (k=10, calibration normalization)

• Gaussian blur sigma=2, convert to frequencies before pixel FlowSOM, normalize including calibration channel
• Pixel hClusters found using z-score cap=3, k=20, and iterative thresh=2 (1 iteration)
• Cell-based clustering using frequencing of 20 hClusters (cap): normalize by size
• Include calibration channel normalization for cell clustering
• k=10 for cell clustering

1 Histogram of counts in pixel clusters

2 Heatmaps of pixel clusters x markers

3 Cell-based FlowSOM clustering using 100 pixel clusters

3.1 Cell cluster x frequency of pixel clusters

3.2 Cell cluster x marker (not weighted, segmentation)

3.3 Cell cluster x marker (weighted by pixel cluster frequency)

3.4 Point breakdown

3.5 Compare frequency with calibration

4 Cell-based FlowSOM clustering using hClusters (cap, k=20)

4.1 Cell cluster x frequency of pixel clusters

4.2 Cell cluster x marker (not weighted, segmentation)

4.3 Cell cluster x marker (weighted by pixel cluster frequency)

4.4 Point breakdown

4.5 Compare frequency with calibration